Les parasites à cycle de vie complexe peuvent modifier le comportement de leurs hôtes intermédiaires,ce qui semble souvent faciliter la transmission du parasite à l'hôte final. La manière dont les parasites y parviennent et, plus précisément, ce qui est modifié dans le cerveau de l'hôte, reste en grande partie à découvrir. Nous avons étudié ici l'épinoche à trois épines (Gasterosteus aculeatus), l'hôte intermédiaire du parasite cestode Schistocephalus solidus. Lorsque infectées, les épinoches présentent une diminution dans leurs réponses antiprédateur. À ce jour, nous savons que les épinoches infectées présentent une augmentation de l'expression cérébrale de leur gène IMPA 1, qui code l'enzyme IMPase1, une étape clé de la synthèse du myo-inositol. Il est intéressant de noter que les niveaux d'IMPase 1 et de myo-inositol sont les principales cibles du traitement au lithium chez les patients atteints de troubles bipolaires. Bien qu'ils soient des candidats prometteurs, nous ne savons pas s'ils sont directement impliqués dans les modifications comportementales chez les poissons infectés. Notre principal objectif était donc d'effectuer une analyse fonctionnelle pour savoir si une altération de la voie cérébrale du myo-inositol avait une implication directe dans ces altérations. Nous avons injecté à des poissons non infectés du myo-inositol exogène (90 mM, n = 20) ou une solution saline (25 ppt, n = 20) pour induire la production de myo-inositol endogène, et nous avons exposé les poissons infectés à du chlorure de lithium (12,5 mM, n = 20). Contrairement à nos attentes, les poissons non infectés exposés à du myo-inositol exogène ou endogène n'ont pas montré d'altération de leur comportement. Cependant, les poissons infectés traités au lithium ont passé moins de temps à nager près de la surface, ont par couru une distance plus courte, ont eu une latence plus élevée pour se nourrir et ont passé plus de temps sans bouger après une attaque de prédateur. Ces résultats suggèrent que la voie du myo-inositol pourrait être impliquée dans les altérations comportementales observées chez les épinoches infectées. Ce mémoire contribue à l'élucidation des mécanismes moléculaires qui sous-tendent l'altération du comportement chez un hôte due à la présence d'un parasite. / Complex life cycle parasites can alter the behavior of their intermediate hosts, which often seems to facilitate the parasite transmission to the final host. How parasites achieve this and, more specifically,what is changed in the host brain remains largely uncovered. Here we studied the threespine stickleback (Gasterosteus aculeatus), the intermediate host of the cestode parasite Schistocephalus solidus. While infected, sticklebacks have severe impairments in their antipredator responses. To date, we know that infected sticklebacks have an increase in their IMPA 1 gene brain expression, which encodes the IMPase 1 enzyme, a key step in the myo-inositol synthesis. Interestingly, IMPase 1 and myo-inositol levels are the main targets of lithium treatment in patients with bipolar disorder. Although promising candidates, we do not know if they are directly implicated in behavioral alterations in Schistocephalus-infected fish. Thus, our main objective was to perform a functional analysis of whether an alteration in the cerebral myo-inositol pathway had a direct implication in such alterations. We injected uninfected fish with exogenous myo-inositol (90 mM, n = 20) or with a saline solution (25 ppt, n = 20) to induce the production of endogenous myo-inositol, and exposed infected fish to lithium chloride (12.5 mM, n= 20). Contrary to our expectations, uninfected fish exposed to exogenous or endogenous myo-inositol did not show alterations in their behavior. However, infected fish treated with lithium spent less times wimming close to the surface, traveled a shorter distance, had a higher latency to feed, and spent more time frozen after a predator attack. These results suggest that the myo-inositol pathway might be implicated in the behavioral alterations observed in infected sticklebacks. This thesis contributes to the elucidation of the molecular mechanisms underlying behavioral alteration in a host due to the presence of a parasite.

Les membres de la famille des 5-phosphatases d’inositols polyphosphates et de phosphoinositides sont des enzymes caractérisées par la présence de deux domaines catalytiques conservés qui hydrolysent un phosphate en position 5 sur un noyau inositol. SKIP (Skeletal Muscle and Kidney enriched Inositol Phosphatase), également appelée Pps (Putative PI 5-phosphatase) est un des derniers membres de la famille des 5-phosphatases à avoir été découvert à ce jour. Cette enzyme hydrolyse majoritairement le phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) et le phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Les phosphoinositides (PtdIns) représentent environ 10% des lipides membranaires et sont impliqués dans de nombreuses cascades de signalisation cellulaire conduisant, entre autres, à la prolifération, l’apoptose, la différenciation, la sécrétion, le trafic vésiculaire et la mobilité cellulaire.<p>Des études de surexpression de SKIP en cellules tendent à montrer que cette protéine pourrait jouer un rôle de régulateur négatif dans la formation du cytosquelette d’actine et/ou dans la voie de signalisation de l’insuline. <p><p>Afin d’étudier in vivo la fonction de la protéine SKIP chez la souris, nous avons décidé de générer des souris transgéniques surexprimant cette protéine de manière conditionnelle. Dans ce but, nous avons infecté des embryons murins par des lentivirus porteurs d’un transgène SKIP et avons obtenu, après réimplantation des embryons infectés dans des femelles pseudogestantes, deux lignées de souris transgéniques. Celles-ci ont ensuite été croisées avec des souris exprimant la recombinase Cre de manière ubiquitaire afin de pouvoir activer la transcription de SKIP dans l’ensemble des organes. Des expériences de Western blot, de dosage d’activité 5-phosphatase ainsi que des PCR en temps réel sont venus confirmer la présence de la protéine transgénique et de son activité catalytique.<p>L’ensemble des expériences qui ont été menées du point de vue phénotypique tend à montrer que dans notre modèle, la surexpression de SKIP ne provoque aucune anomalie évidente du point de vue anatomique, glycémique ou immunologique. Toutefois, des expériences concernant la physiologie rénale ont été réalisées sur base des résultats d’immunohistochimie et nous ont permis de détecter une anomalie dans les mécanismes de réabsorption d’eau ainsi que dans l’expression et la phosphorylation des canaux hydriques AQP2.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Inositolpyrophosphate (PP-InsPs) sind eine wichtige Gruppe eukaryotischer Botenstoffe, die mit verschiedenen Prozessen wie Apoptose, Phosphathomeostase und Insulinsignalkaskaden verknüpft sind. Trotz ihrer Entdeckung vor mehr als 20 Jahren bleibt es eine Herausforderung, die Signalmechanismen dieser Moleküle zu verstehen. Ursachen dafür sind der limitierte Zugang zu synthetischen PP-InsPs und ein Mangel an allgemein zugänglichen analytischen Methoden. Daher wurden in dieser Arbeit chemische und analytische Verfahren entwickelt, um unser Verständnis von diesen Molekülen sowohl auf ein biochemischer als auch auf zelluläre Ebene zu verbessern. Um der Knappheit an synthetischen PP-InsPs entgegen zu wirken, wurde eine hocheffiziente chemoenzymatische Synthese entwickelt, bei der mehr als 100 mg aller wesentlichen PP-InsPs aus Säugern hergestellt werden konnten. Parallel wurde ein neues analytisches Werkzeug entwickelt, dass Konzentrationen von PP-InsPs in komplexen Proben quantifizieren konnte. Mittels Enzymkatalyse konnten 13C-markiertes myo-inositol und 13C-markierte PP-InsPs hergestellt werden und niedrige Konzentrationen mit nuklearer Magnetresonanzspektroskopie detektiert werden. In vitro waren diese Verbindungen sehr nützlich, um PP-InsP Kinasen von Pflanzen und Säugern zu charakterisieren. Endogene Konzentrationen von PP-InsPs konnten durch metabolisches Markieren mit 13C-markiertem myo-inositol in humanen Zelllinien quantifiziert werden. Letztendlich wurde mittels eines neuen entwickelten proteomischen Ansatzes endogene Proteinpyrophosphorilierung, eine von PP-InsP eingebaute posttranslationale Proteinmodifikation, in menschlichen Zelllinien zum ersten Mal nachgewiesen. Zusammenfassend haben die aufgelisteten chemischen und analytischen Werkzeuge ein hohes Potenzial unser Verständnis der Signalmechanismen hinter den diversen Phänotypen der PP-InsPs zu stärken und Forschungsarbeit in dieser Richtung zu beschleunigen. / Inositol pyrophosphates (PP-InsPs) are an important group of second messengers that intersect with a wide range of processes in eukaryotic cells including phosphate homeostasis, insulin signaling and apoptosis. Despite their discovery more than two decades ago, elucidating the underlying signaling mechanisms remains a significant challenge. Therefore, a new set of chemical and analytical methods was developed here to improve our understanding of these intriguing molecules on the biochemical and cellular level. To overcome the shortage of synthetic PP-InsPs, a highly efficient and scalable chemoenzymatic approach was designed and the major mammalian PP-InsPs could be obtained in hundreds of milligram quantities and in high purity. In parallel, a new analytical tool was developed to quantify levels of PP-InsPs in complex samples. Chemoenzymatic access to 13C-labeled myo-inositol and 13C-labeled PP-InsPs enabled the detection of low concentrations of PP-InsPs using nuclear magnetic resonance spectroscopy. In vitro, these compounds were of great use for the biochemical characterization of PP-InsPs kinases from mammals and plants. Endogenous pools of PP-InsPs from human cell lines were identified and quantified by metabolic labeling with 13C-labeled myo-inositol. Finally, a new proteomics workflow towards the detection of protein pyrophosphorylation, a posttranslational modification mediated by PP-InsPs, using mass spectrometry was optimized and endogenously modified mammalian proteins could be identified for the first time and with high confidence. Taken together, the chemical and analytical tools presented here have great potential to accelerate the understanding of PP-InsP signaling and metabolism. Access to large amounts of PP-InsPs together with a reliable quantification method and the detection of endogenous protein pyrophosphorylation sites will be essential to unravel the signaling mechanisms underlying the diverse phenotypes associated with these metabolites.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Actively proliferating cells constantly monitor and re-adjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae, multiple studies have suggested that lysine acetylation has a role in coordinating phospholipid metabolism, yet its contribution towards phospholipid homeostasis remains uncharacterized. In this study we undertook a genetic screen to explore the connection between lysine acetylation and phospholipid homeostasis. We found that mutants of the lysine acetyltransferase complex, NuA4, shared a negative genetic interaction with a mutant of Sec14, a lipid-binding protein that regulates Golgi phospholipid composition. Through transcriptome, genetic, cell biology, and chemical analysis, we discovered that the growth defects between NuA4 and Sec14 mutants is likely derived from impaired fatty acid biosynthesis suggesting a role for NuA4 as a positive regulator of fatty acid biosynthesis. Secondly, we discovered that acetylation on the conserved lysine residue K109 inhibits the localization and function of the Oxysterol-Binding Protein Osh4- a lipid-binding protein that antagonizes the function of Sec14 at the Golgi. Furthermore, regulation of Oxysterol-Binding Proteins by acetylation may be a conserved mechanism as we found that Osh1, a homologue of Osh4, was also acetylated on the equivalent lysine residue. Altogether, we have demonstrated that lysine acetylation can target multiple different phospholipid metabolic pathways which implies that it has a very important role for the regulation of phospholipid homeostasis.

In the context of fetal alcohol spectrum disorders, we investigated how the nitric oxide (NO) signaling pathway influences intracellular calcium (Ca2+) to mediate alcohol resistance, using a primary cell culture model of cerebellar granule neurons (CGN). Alcohol during fetal brain development triggers abnormally high apoptotic cell death in vulnerable neuronal populations, culminating in serious behavioral and cognitive deficits that persist into adulthood. Prior studies demonstrated that the NO signaling pathway [neuronal nitric oxide synthase → NO → soluble guanylyl cyclase → cyclic guanosine monophosphate → protein kinase G (PKG)] mitigates alcohol toxicity, consequently diminishing neuronal loss both in vivo and in vitro. Endoplasmic reticulum (ER) Ca2+ release, a key apoptotic mechanism, requires the inositol 1,4,5-trisphosphate receptor (IP3R), a known PKG substrate. Our studies focused on this crucial intersection point where the NO signaling cascade can influence Ca2+-mediated apoptotic mechanisms, and exposed a downstream mechanism where NO can moderate alcohol neurotoxicity. We hypothesized that as alcohol disturbs neuronal Ca2+ homeostasis to trigger cell death, the NO signaling pathway counters it by limiting Ca2+ release from the ER. We examined first the role of the phospholipase C (PLC) pathway [PLC → inositol 1,4,5-trisphosphate → IP3R → Ca2+] in developmental neurotoxicity through our in vitro CGN model, extending previous in vivo studies. We found that alcohol terminates developing neurons by eliciting abnormal Ca2+ release from the ER rather than from an extracellular source, via a PLC - IP3R-dependent signaling mechanism. Inhibiting either calcineurin or Ca2+ / calmodulin-dependent protein kinase ii (CaMKii), which participate in parallel Ca2+-activated apoptotic cascades, shielded CGN cultures from alcohol. Blocking the mitochondrial Ca2+ uniporter or the mitochondrial permeability transition pore also provided neuroprotection. That the activated pathways must interact to generate cell death likely explains why inhibiting one of multiple parallel signaling cascades limits alcohol toxicity. We next demonstrated that activating the NO pathway downstream at PKG eliminated both alcohol-related neuronal death and the accompanying rapid rise in intracellular Ca2+, an effect that markedly resembled IP3R inhibition. Experiments that temporally manipulated the addition of PKG activators in relation to alcohol exposure linked PKG's obstruction of alcohol-induced Ca2+ elevations to alcohol resistance. In contrast, brain-derived neurotrophic factor (BDNF), which does not rely on PKG to provide neuroprotection, failed to block alcohol-induced Ca2+ elevations while preventing alcohol toxicity. This indicates that although PKG blocks alcohol-induced Ca2+ elevations, averting these Ca2+ elevations is not necessary for neuroprotection. BDNF may confer alcohol resistance through an as yet unidentified process downstream from the disruption of intracellular Ca2+. In summary, we established that 1) alcohol induces toxic Ca2+ elevations originating from the ER through a PLC - IP3R-dependent pathway, and that 2) PKG-mediated alcohol resistance is linked to preventing the intracellular Ca2+ surges. These findings support the hypothesis that the NO signaling pathway shields developing neurons from alcohol by limiting Ca2+ release from the ER.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

The brain is a major target site in acute cyanide intoxication, as indicated by several symptoms and signs. Cyanide inhibits the enzyme cytochrome oxidase. This inhibition causes impaired oxygen utilization in all cells affected, severe metabolic acidosis and inhibited production of energy. In this thesis, some neurotoxic effects of cyanide, in particular, the effects on dopaminergic pathways were studied. In a previous study, decreased levels of striatal dopamine and HVA were found after severe cyanide intoxication (5-20 mg/kg i.p.). However, increased striatal dopamine were found in rats showing convulsions after infusion of low doses of cyanide (0.9 mg/kg i.v.), at the optimal dose rate (the dose rate that gives the treshold dose). Increased striatal dopamine synthesis was observed in rats after cyanide treatment and in vitro. Furthermore, in rat, as well as in pig striatal tissue, cyanide dose- dependently increased the oxidative deamination of 5-HT (MAO-A) and DA (MAO-A and -B) but not that of PEA (MAO-B). Thus cyanide affects both the synthesis and metabolism of dopamine. In rats, sodium cyanide (2.0 mg/kg, i.p.) decreased the striatal dopamine Dj- and D2-receptor binding 1 hour after injection. Increased extracellular levels of striatal dopamine and homovanillic acid were also shown after cyanide (2.0 mg/kg; i.p.). DOPAC and 5-HIAA were slightly decreased. This indicates an increased release or an extracellular leakage of dopamine due to neuronal damage caused by cyanide. Thus the effects of cyanide on dopamine Dj- and D2~receptors could in part be due to cyanide-induced release of dopamine. Because of reported changes in intracellular calcium in cyanide-treated animals, the effects of cyanide on inositol phospholipid breakdown was studied. Cyanide seemed not to affect the inositol phospholipid breakdown in vitro. The effects of cyanide on the synthesis and metabolism of brain GAB A were also examined. A decreased activity of both GAD and GAB A-T were found in the rat brain tissue. The reduced activity of GAB A-T, but not that of GAD returned to the control value after adding PLP in the incubation media. The cyanide-produced reduction of GABA levels will increase the susceptibility to convulsions, and could partly be due to GAD inhibition. In conclusion, cyanide affects the central nervous system in a complex manner. Some effects are probably direct. The main part, however, appears to be secondary, e.g. hypoxia, seizures, changes in calcium levels or transmitter release produced by cyanide. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 7 uppsatser</p> / digitalisering@umu

Cell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.